Effect of liver transplants with retrograde reperfusion on early postoperative recovery of liver function and its risk factors

Background The purpose of this study was to investigate effect of liver Transplants (LT) with retrograde reperfusion on early postoperative recovery of liver function and its risk factors. Methods We conducted a retrospective analysis of clinical data from 136 liver transplantation (LT) patients at the 900th Hospital of the Chinese People’s Liberation Army Joint Support Army, covering the period from January 2015 to January 2021. All participants provided informed consent, adhering to medical ethics guidelines. Patients were stratified into two groups based on the liver perfusion technique used: retrograde reperfusion (RTR, n = 108) and initial portal reperfusion (IPR, n = 28). Our study focused on a subset of 23 patients from each group to compare postoperative liver function recovery. The final analysis included 86 RTR and 28 IPR cases after excluding 8 RTR patients who underwent initial hepatic artery reperfusion and 14 who received simultaneous hepatic artery and portal vein reperfusion. Further subdivision within the RTR group identified 19 patients with early hepatic allograft dysfunction (EAD) and 67 without, allowing for an assessment of the influence of preoperative and intraoperative parameters, as well as perfusion methods, on EAD incidence post-LT. Results Alanine aminotransferase (ALT) was 329 (211 ~ 548) and 176 (98 ~ 282) U/L on the 3rd and 7th day after RTR, respectively, which was significantly lower than 451 (288 ~ 918) and 251 (147 ~ 430) U/L in the IPR group (Z =-1.979, -2.299, P = 0.048, 0.021). Aspartate aminotransferase (AST) on postoperative days 3, 5, and 7 was 252 (193, 522), 105 (79, 163), and 93 (41, 135) U/L in the RTR group, respectively; it was also significantly lower than 328 (251, 724), 179 (129, 306), and 150 (91, 200)U/L in the IPR group (Z=-2.212, -3.221, -2.979; P = 0.027, 0.001, 0.003). Logistic regression analysis showed that MELD score was an independent risk factor for EAD after LT. Conclusion RTR LT is more favorable for patients’ early postoperative liver function recovery. For patients undergoing LT for RTR, preoperative MELD score was an independent risk factor for their postoperative development of EAD. Supplementary Information The online version contains supplementary material available at 10.1186/s12893-024-02467-3.


Introduction
At present, liver Transplants (LT) has become the most effective treatment for end-stage liver disease [1].After mortality, various organs, particularly the liver, encounter systemic inflammatory responses, hemodynamic fluctuations, and endocrine disturbances.These factors collectively contribute to early onset of ischemiareperfusion injury (IRI) in the liver.Consequently, such pathophysiological alterations in the liver are inevitable, potentially leading to higher mortality rates and a greater likelihood of complications following recipient surgery [2][3][4].According to some studies, retrograde reperfusion technique (RTR) can effectively improve the early recovery of liver function after LT [5,6].At present, there are not many studies on the effect of RTR on early postoperative liver function recovery after LT, and there is a lack of studies on the analysis of risk factors associated with early postoperative liver allograft dysfunction (EAD) in patients with RTR.Hence, this investigation scrutinized the impact of RTR on the expeditious convalescence of hepatic function subsequent to transplantation-a factor acknowledged for its association with early allograft dysfunction (EAD).Such insights serve to guide clinicians in making judicious therapeutic determinations.

Patients and methods
We retrospectively reviewed the clinical data of 136 patients who underwent liver transplantation (LT) at the 900th Hospital of the Chinese People's Liberation Army Joint Support Army from January 2015 to January 2021.Following a 1:1 propensity score matching, patients were evenly divided into the retrograde reperfusion (RTR) group and initial portal reperfusion (IPR) group, each consisting of 23 individuals.For the methodology details, refer to Supplementary Fig. 1.Subsequent analysis focused on comparing postoperative liver function recovery between the two groups.Patients in the RTR group were further categorized based on the occurrence of early hepatic allograft dysfunction (EAD) into EAD and non-EAD subgroups.We examined the influence of preoperative and intraoperative factors, as well as the liver perfusion technique employed during transplantation, on the incidence of EAD post-LT.
The diagnostic criteria for EAD were as follows: (1) TB ≥ 10 g/L (171 mmol/L) at 7 days after transplantation; (2) INR ≥ 1.6 at 7 days after transplantation; and (3) ALT or AST > 2,000 U/L within 7 days after transplantation.EAD can be diagnosed if one or more of the above conditions are met [2].
Each patient provided informed consent, approved by the hospital's ethics committee in accordance with medical ethical standards.All protocols adhered to pertinent guidelines, with written consent obtained following the Declaration of Helsinki principles.

Data acquisition
All data in this study were collected from the electronic medical records and anesthesia system of each subject.The indicators were obtained from the blood test results of the patient before surgery and the postoperative indicators were obtained from the blood test results of the patient on Day 1, 3, 5, 7 and 14.

Surgical methods
The donor liver was prepared using a cold storage solution, typically the University of Wisconsin (UW) solution, prior to the transplant procedure.We have now included detailed characteristics of the donors and the grafts used in the transplants, recognizing their potential impact on the outcomes of liver transplantation.These characteristics encompass donor age, donor body mass index (BMI), degree of graft steatosis, and functional warm ischemia time.Specifically, the average donor age was 45 years, with a BMI range of 22 to 30 kg/m2.Graft steatosis was assessed and categorized as none (0%), mild (1-30%), moderate (31-60%), and severe (> 60%).Functional warm ischemia time, defined as the duration from graft retrieval to blood reperfusion, averaged 35 min.These factors were rigorously documented and analyzed for their correlation with the incidence of EAD post-transplantation.The operation commenced under general anesthesia with a bilateral subcostal incision providing access.During the procedure, 800 ml of fresh frozen plasma was continuously infused into the portal vein.In the case of retrograde perfusion, the orifices of the upper and lower inferior vena cava (IVC) were opened post-anastomosis, allowing for reversible liver perfusion with a fraction of the returning blood flow.The diseased liver was excised, and the donor liver was implanted.Vascular anastomoses for the hepatic veins and the portal vein were completed, followed by arterial reconstruction.
Bile duct reconstruction was tailored to patient-specific anatomical requirements [7].Intraoperative management included hemodynamic monitoring and administration of methylprednisolone, starting at a dose of 30 mg/ day and adjusted based on liver function and blood parameters.
In the allocation of liver transplantation techniques, patients were not randomly assigned to the retrograde reperfusion (RTR) and initial portal reperfusion (IPR, also known as antegrade reperfusion) groups.Instead, the selection was based on specific intraoperative assessments and preoperative conditions.Retrograde reperfusion was predominantly chosen for patients exhibiting certain clinical parameters that indicate a higher risk of postoperative complications with traditional antegrade reperfusion.These parameters included, but were not limited to, pre-existing vascular abnormalities, anticipated complications from prolonged cold ischemia times, and the surgeon's anticipation of hemodynamic instability during the procedure.This approach was intended to minimize risks associated with portal congestion and improve postoperative outcomes by ensuring optimal blood flow management during the critical reperfusion phase.
This steroid regimen was tapered over three months post-surgery, guided by liver function tests.

Postoperative management and immunosuppressive regimen
All patients were given intravenous drip of 500 mg methylprednisolone during surgery and tacrolimus + mycophenolate mofetil + Methylprednisolone regimen after transplantation.Tacrolimus was used according to renal function from 1 to 3 d after surgery, and the tacrolimus drug concentration was controlled at 8 to 12 mg/L in the early postoperative period.
Methylprednisolone was initiated at 300 mg intravenously every 24 h.Methylprednisolone was administered at a dose decrement of 30 mg/d according to postoperative liver function and blood routine parameters.Intravenous methylprednisolone was changed to oral (20 mg/ day) after 1 month, and steroids were gradually stopped within 3 months after surgery according to follow-up parameters.Since patients who underwent incidental appendectomies were also going to use immunosuppressive drugs during the postoperative period, each appendix stump was ligated, and transposition sutures were made, using polypropylene sutures, to avoid stump failures [8].

Statistical analysis
SPSS 26.0 statistical software was used for data analysis.The age, BMI and other data in accordance with the normal distribution in the two groups were expressed as x ± ‾s, and the independent sample t-test was used for comparison; those not in accordance with the normal distribution were expressed as median (Q1, Q3), and the rank sum test was used for comparison.1:1 propensity score matching (PSM) was used to balance confounding bias factors.Logistics regression was used to analyze the independent influencing factors of EAD after LT and draw ROC curves.P < 0.05 was considered statistically significant.

Patient 's clinical information
Among 136 patients with LT, they were divided into IPR group (n = 108) and IPR group (n = 28) according to the order of intraoperative reflow.After applying inclusion criteria, 24 patients were excluded.A total of 114 patients were finally included in this study, including 86 patients (75.43%) with RTR and 28 patients (24.57%) with IPR.The integrated preoperative baseline data, including age, MELD score, BMI, intraoperative factors, liver function indicators, coagulation markers, and donor factors.To assess intraoperative reflow's impact on postoperative liver function, these factors were balanced using propensity score matching (PSM) for comparable patient selection.

Analysis of matching data of patients in RTR and IPR group before PSM matching
Before PSM matching, some indicators of RTR, including age, albumin (ALB), international standardization ratio (INR), total bilirubin (TB), total operative time (TOT), total blood loss, and alanine aminotransferase (AST), were significantly different from IPR group (P < 0.05).(Table 1)

Analysis of matching data of patients in RTR and IPR group after PSM matching
86 patients in the RTR group and 28 patients in the IPR group included in the study were paired in a 1:1 manner using the PSM matching method, and the matching tolerance was set to 0.2.The factors that were significantly different between the RTR and IPR groups before matching were: age, ALB, INR, MELD score, total operation time, intraoperative blood loss, TBil, and AST.SPSS was used for matching to select patients with successful matching.Ultimately, 23 patients who underwent liver transplantation (LT) prior to adopting retrograde reperfusion (RTR) were included in the study.After propensity score matching, no significant differences were observed in preoperative and intraoperative indicators between the RTR and initial portal reperfusion (IPR) groups (P < 0.05), as detailed in Table 2.

Postoperative liver function recovery after PSM matching in RTR group and IPR group
Non-parametric tests of two independent samples were performed for liver function on days 1, 3, 5, 7, and 14 in patients after LT in the RTR group and IPR group.There were significant differences in serum ALT, AST and TBil concentrations between the two groups after surgery.Postoperative ALT was lower in the serum of patients in the RTR group at day 3 (P = 0.027) and day 7 (P = 0.030).Compared with postoperative serum AST in IPR group, there were significant differences in serum AST concentration between the two groups on Day 3, Day 5 and Day 7. TBil in the IPR group was significantly higher than RTR group on postoperative d5, but the overall trend showed that the postoperative TBil change trend tended to be the same in both groups.(Table 3).

Logistics analysis of EAD after RTR LT
According to Table 3, patients who underwent RTR had faster recovery of liver function after transplantation, so patients in the RTR group (86) were further divided into the EAD group (n = 19) and the non-EAD group (n = 67).The risk factors affecting EAD after transplantation were analyzed from three aspects: donor liver factors, intraoperative factors and recipient factors.In univariate analysis, we found that CIT, PT, Tbil and MELD scores were risk factors for postoperative EAD, so we further included these four variables in multivariate analysis, and finally concluded that MELD score was an independent risk factor for postoperative EAD in patients.(P < 0.05, Table 4).

ROC curve analysis results
The preoperative MELD score of the patients was analyzed by ROC curve, and finally the area under the ROC curve (AUC) of the MELD score for predicting postoperative EAD was 0.678, and the Youden index was 0.352 by sensitivity and specificity calculation.The cut-off value of the preoperative MELD score was 25.5 (Fig. 1).

Discussion
LT holds promise for patients with end-stage liver disease.Since the application of DCD donors, the incidence of EAD after LT has remained high.The preservation of graft function faces great challenges before donor acquisition, during storage, and during implantation.At present, studies on IRI in the organ suggest that both ischemic and reperfusion phases can cause damage to donor function [9].Due to the effect of IRI, impaired ATP synthesis in mitochondria is impaired, and damaged intracellular material reaches lysosomes for degradation process as autophagy.Autophagy can remove dysfunctional mitochondria in vivo to ensure normal functioning of cells [10,11].Autophagic activity is therefore a protective factor in alleviating IRI injury.Cheng's team pioneered the construction of a retrograde perfusion rat model and compared the effect of reflow mode on postoperative liver function by measuring the attenuation intensity of autophagic activity, and concluded that the duration of autophagic activity in RTR protects  the function of healthy mitochondria [12].RTR was pioneered by Kniepeiss's [5] team, and the inverse perfusion method showed irreplaceable advantages in reducing transaminases.Our study showed that ALT at 3 and 7 days after transplantation was significantly lower in patients in the reverse perfusion group than in the positive perfusion group (P < 0.05).There was significant difference in AST at 3, 5 and 7 d after operation in the reverse perfusion group (P < 0.05).ALT and AST gradually decreased and tended to be stable within 14 d after operation.The change trend of liver function was similar to the change trend of autophagy ability in the above animal experiments.It is suggested that the recovery of ALT and AST after transvenous retrograde perfusion LT has a positive effect.Transvenous reverse perfusion LT is not the mainstream choice in international organ transplantation centers.Heidenhain et al. [13] conducted a retrospective study of 131 patients with orthotopic LT (66 of whom were treated with simultaneous forward reperfusion of hepatic artery and portal vein, and 65 with retrograde reperfusion of vein) and found that the incidence of postoperative IPF in patients who underwent retrograde reperfusion of vein (13.4%) was significantly lower than that in patients who underwent forward perfusion (31.3%), and the difference had statistical significance (P = 0.022).The incidence of EAD in this study was about 22%, suggesting that the reverse perfusion method has some promise in reducing the risk of liver dysfunction in transplantation.Although the reverse perfusion method helps to reduce the incidence of EAD after LT, the proportion of EAD still remains large.Postoperative EAD is a common complication after LT and is often induced by a variety of factors.The terminal stage of recipient liver function is often accompanied by coagulation abnormalities, which are generally characterized by prolonged PT, increased intraoperative blood loss, and increased need for the use of blood products [14].Relevant reports have found that for every unit increase in red blood cells used during surgery, the incidence of postoperative EAD in recipients increases by 8% [15].
Prolonged cold ischemia time leads to a significant increase in the incidence of nonfunction of the recipient transplanted liver [16], and cold ischemia time is also associated with longer post-transplant hospital stay, higher incidence of primary graft nonfunction, and hyperbilirubinemia [17].Sibulesky et al. [18] analyzed 350 donor cold ischemia time and divided the patients into three groups according to the time, the cold ischemia time was less than 8 h (48%), 8 to 12 h (38%), and more than 12 h (14%) groups, and their 1-year graft survival rates after surgery were 92%, 94%, and 87%, respectively, and it was considered that prolonged cold ischemia time would lead to early graft dysfunction.Univariate analysis in this study revealed that cold ischemia time did not reach statistical significance.The donors for donation after cardiac death (DCD) in our institution originated from designated regions, with all liver transplant donors sourced from specific cities within our province, ensuring that cold ischemia times did not exceed 12 h and showed minimal variation.Although cold ischemia time did not  emerge as an independent risk factor in our analysis, these findings underscore the importance of minimizing cold ischemia times to optimize donor liver function preservation.
The MELD score was initially used as a predictor of survival in end-stage liver disease [19].A study report showed that preoperative recipient MEDL score was positively correlated with the incidence of EAD after LT [20].The results of this study showed that MELD score greater than 25.5 was an independent risk factor for postoperative EAD in transplant patients, but the predictive ability of MELD score for abnormal liver enzymes or EAD diagnosed due to TB abnormalities was different.This study classified EAD patients with ALT or AST > 2,000 U/L as type A, patients with TB > 171µmol/L or INR > 1.6 at 7 days after surgery as type B, and patients with type B had a risk of postoperative death.However, the predictive value of preoperative MELD score for postoperative EAD in type B patients [21].

Conclusion
In summary, retrograde reperfusion in liver transplantation (LT) enhances early postoperative liver function recovery.The preoperative Model for End-Stage Liver Disease (MELD) score independently predicts the risk of early hepatic allograft dysfunction (EAD) in recipients undergoing retrograde reperfusion.Strategies aimed at improving preoperative tolerance and lowering the MELD score may mitigate EAD risk.However, the limited number of EAD cases in our study necessitates larger randomized trials to validate the predictive reliability of the MELD score for postoperative EAD.

Table 1
Comparison of preoperative and intraoperative clinical data between the two groups of RTR group and IPR group before PSM matching Abbreviations: HDT, Hepatic devascularization time; BMI, Body Mass Index; ALB, Albumin; CIT, Cold ischemia time; MELD, Model for end-stage liver disease; INR, international standardization ratio; Tbil, total bilirubin; TOT, total operative time; ALT, alanine aminotransferase; ALP, alkaline phosphatase; GGT, Glutamyl transpeptidase; AST, aspartate transaminase;

Table 2
Comparison of preoperative and intraoperative clinical data between the two groups of RTR group and IPR group after PSM matching

Table 3
Postoperative recovery of liver function in RTR and IPR group